Potyviral NIa Proteinase, a Proteinase with Novel Deoxyribonuclease Activity

Abstract

The NIa proteinase from pepper vein banding virus (PVBV) is a sequence-specific proteinase required for processing of viral polyprotein in the cytoplasm. It accumulates in the nucleus of the infected plant cell and forms inclusion bodies. The function of this protein in the nucleus is not clear. The purified recombinant NIa proteinase was active, and the mutation of the catalytic residues His-46, Asp-81, and Cys-151 resulted in complete loss of activity. Most interesting, the PVBV NIa proteinase exhibited previously unidentified activity, namely nonspecific double-stranded DNA degradation. This DNase activity of the NIa proteinase showed an absolute requirement for Mg 2؉ . Site-specific mutational analysis showed that of the three catalytic residues, Asp-81 was the crucial residue for DNase activity. Mutation of His-46 and Cys-151 had no effect on the DNase activity, whereas mutant D81N was partially active, and D81G was completely inactive. Based on kinetic analysis and molecular modeling, a metal ion-dependent catalysis similar to that observed in other nonspecific DNases is proposed. Similar results were obtained with glutathione S-transferase-fused PVBV NIa proteinase and tobacco etch virus NIa proteinase, confirming that the DNase function is an intrinsic property of potyviral NIa proteinase. The NIa protein present in the infected plant nuclear extract also showed the proteinase and the DNase activities, suggesting that the PVBV NIa protein that accumulates in the nucleus late in the infection cycle might serve to degrade the host DNA. Thus the dual function of the NIa proteinase could play an important role in the life cycle of the virus.

Visualization of the Interaction between the Precursors of VPg, the Viral Protein Linked to the Genome ofTurnip Mosaic Virus, and the Translation Eukaryotic Initiation Factor iso 4E In Planta

“…Consequently, nuclear/nucleolar interaction of VPg-Pro with eIF(iso)4E may perturb the nuclear role of the factor by sequestration. Interestingly, VPg-Pro possesses DNase and RNase activities (1,8). Association with eIF(iso)4E might thus target VPg-Pro to particularly sensitive areas for DNA and RNA degradation.…”

Sharka: The Past, The Present and The Future

“…The active – catalytic – sites of NIa-Pro are defined by a consensus heptapeptide sequence surrounding each cleavage site containing triad of His-Asp-Cys, which are conserved among the potyviruses [70]. The mutation of the catalytic residues His46, Asp81, and Cys151 resulted in complete loss of activity [71]. In addition, NIa proteinase possesses structural motifs shared with cellular serine proteases with the substitution of serine by a cysteine as the active site nucleophile.…”

Unfolding the secrets of plum pox virus: from epidemiology to genomics

“…Interaction with viral replicase (NIb) and non-specific binding of RNA also implies the involvement of NIa-pro in the replication process (Guo et al, 2001;Daros and Carrington, 1997). Nuclear localization of NIa-pro in the late infection stage together with demonstrated deoxyribonuclease activity could imply its action in degradation of the host DNA (Anindya and Savithri, 2004).…”